JP7621107B2 - Cholecalciferol sulfate and its use for the treatment of vitamin D deficiency - Google Patents

Description

The present invention relates to the use of pharma- ceutically acceptable cholecalciferol sulfate for combating vitamin _D3 deficiency in vertebrates, particularly humans, by an administration method in which at least 5% of the cholecalciferol sulfate is delivered from the administration site to the systemic fluid transport system of the human body without metabolism as such, to new pharma-ceutically acceptable cholecalciferol sulfates, and to pharmaceutical compositions for the above-described administration in dosage forms containing pharma-ceutically acceptable cholecalciferol sulfate.

It is known that a large proportion of the world's population suffers from vitamin _D3 deficiency. It is known that under natural conditions, vitamin _D3 is produced in humans as water-soluble vitamin _D3 sulfate (cholecalciferol sulfate) from 7-dehydrocholesterol by exposure of the skin to UV radiation. Many people, especially in the winter season, are unable to expose themselves to UV radiation sufficiently to form sufficient amounts of vitamin _D3 sulfate in the skin.

Cholecalciferol sulfate is found in breast milk and in small amounts in other types of milk. Some other types of foods, such as cod liver oil, contain the non-sulfated fat-soluble vitamin _D3 (cholecalciferol). In dietary supplements, vitamin _D3 is also found as fat-soluble cholecalciferol. This latter form of vitamin _D3 is not sulfated in the body.

Remington, The Science and Practice of Pharmacy, edited by Allen, Loyd V. Jr., 22nd edition, Pharmaceutical Press. L. E. Reeve et al., The Journal of Biological Chemistry (1981), Vol. 256, No. 2, p. 824

The object of the present invention was to provide the body with natural cholecalciferol sulfate in a rapid or otherwise long-term manner without the need to expose the skin to UV radiation.

The present invention therefore relates to the use of pharma- ceutically acceptable cholecalciferol sulfate for combating vitamin _D3 deficiency in vertebrates, particularly humans, by an administration method in which at least 5% of the cholecalciferol sulfate reaches the systemic fluid transport system of the human body by itself, without metabolism, from the administration site.

The present invention further relates to novel pharma- ceutically acceptable cholecalciferol sulfates, in particular magnesium cholecalciferol sulfate, calcium cholecalciferol sulfate, and L-lysine-cholecalciferol sulfate.

Finally, the present invention relates to a pharmaceutical composition comprising at least one pharma- ceutically acceptable cholecalciferol sulfate and a carrier suitable for transdermal or transmucosal administration, or for intradermal, subcutaneous or intramuscular injection.

FIG. 1 shows the NMR spectrum (200 MHz; internal standard: TMS) of cholecalciferol sulfate L-lysine in D ₄ -methanol.

Surprisingly, it has been found that cholecalciferol sulfate, and therefore vitamin _D3 in its natural form, can be supplied to the body in such a way that they are not immediately metabolized to water-insoluble cholecalciferol or (25OH)-cholecalciferol. The lymphatic system can distribute the water-soluble cholecalciferol sulfate by itself in the body. Water-soluble cholecalciferol sulfate is of course also soluble in blood. However, they can potentially undergo rapid, e.g. enzymatic, degradation.

As used herein, combating vitamin _D3 deficiency means the prevention and treatment of vitamin _D3 deficiency.

Pharmaceutically acceptable cholecalciferol sulfate herein means that the cations of these salts are not toxic to the human body in any way. Pharmaceutically acceptable cations are, for example, Na, Mg, Ca, Zn, ammonium, and protonated amino acids such as protonated lysine.

Cholecalciferol sulfates are usually soluble in water or aqueous alcohol mixtures. Examples of pharma- ceutically acceptable cholecalciferol sulfates with inorganic cations are cholecalciferol sodium sulfate, cholecalciferol magnesium sulfate, cholecalciferol calcium sulfate, and cholecalciferol ammonium sulfate. Examples of pharma-ceutically acceptable cholecalciferol sulfates with organic cations are cholecalciferol trimethylammonium sulfate and cholecalciferol L-lysine sulfate.

The administration method according to the invention is selected such that at least 5% of the administered amount of cholecalciferol sulfate is transported from the administration site to the systemic fluid transport system of the human body without metabolism. Depending on the administration method, preferably at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the administered cholecalciferol sulfate reaches the systemic fluid transport system of the human body from the administration site by itself without undergoing metabolism.

Preferably, less than 70%, preferably less than 60%, e.g., less than 50%, 40%, 30%, 20%, or even less than 10%, or less of the administered cholecalciferol sulfate is chemically modified (metabolized) in at least one of the systemic fluid transport systems selected from blood and lymph over a period of at least 5 minutes, preferably at least 10, 15, 20, or even more than 30 minutes.

The systemic fluid transport systems of the human body are, for example, blood and preferably lymph.

Preferred modes of administration of pharma- ceutically acceptable cholecalciferol sulfate are transdermal or transmucosal administration, as well as intradermal, subcutaneous and intramuscular injections, depot injections including sustained release of the active substance. Oral administration (i.e., delivered to the stomach by swallowing), intravenous and intraarterial injections are expressly excluded as modes of administration.

The molar amount of cholecalciferol sulfate administered generally corresponds to the molar amount recommended for cholecalciferol.

Cholecalciferol sulfate can be prepared by reacting commercially available cholecalciferol with pyridine-sulfur trioxide complex in pyridine and subsequent reaction with triethylamine to give triethylammonium cholecalciferol sulfate, which can then be precipitated in aqueous solution by adding saturated solutions of the appropriate cations to form salts with these cations.

Alternatively, cholecalciferol can be reacted with pyridine-sulfur trioxide complex in pyridine and then reacted directly with the desired cation, optionally in the presence of a suitable buffer, to form the desired salt.

The pharmaceutical compositions according to the invention comprise a suitable carrier or vehicle. These may also contain additional active agents, such as other vitamins, minerals, and trace elements, as well as any type of medicine.

Vehicles or carriers suitable for transdermal administration are all vehicles known for this purpose to those skilled in the art of pharmacy. These include liquid or solid vehicles on a fat or oil base and vehicles on an aqueous or aqueous alcohol base. The formulations can take the form of ointments and oils, lotions, solutions adapted to be sprayed, suspensions and emulsions of any kind, as well as patches containing the active substance in a vehicle. The formulations may contain penetration enhancers such as dimethylsulfoxide, emulsifiers, and any further additives commonly used in pharmacy, such as excipients, flavorings, solubilizers, lubricants, suspending agents, binders, and preservatives.

Formulations suitable for transmucosal administration are, for example, aqueous or alcoholic solutions which may contain further additives, fatty-based suppositories, creams, gels, pastes, foams or sprays, as well as pessaries and tampons suitable for vaginal administration, and solid dosage forms suitable for local administration in the mouth or sublingually, including lozenges containing the active ingredient in a flavoured base, usually sucrose and gum acacia or tragacanth; pastilles containing the active ingredient in an inert base such as gelatin or glycerol; and chewing gum. Formulations for transmucosal administration may also contain penetration enhancers such as dimethylsulfoxide, emulsifiers and any further additives common in pharmacy.

Liquid form formulations for parenteral administration by intradermal, subcutaneous and intramuscular injection include suspensions, solutions or emulsions in oily or aqueous vehicles. The formulations may contain formulatory additives such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for mixing with a suitable vehicle, such as sterile pyrogen-free water, for constitution before use.

A detailed presentation of dosage forms suitable for use in the present invention can be found, for example, in Remington, The Science and Practice of Pharmacy, edited by Allen, Loyd V. Jr., 22nd Edition, Pharmaceutical Press.

Example 1
Preparation of cholecalciferol triethylammonium sulfate 6.5 ml of pyridine was added under nitrogen atmosphere to 1.21 g of pyridine-sulfur trioxide complex (approximately 6.76 mmol of pyridine complexed with SO ₃ ; Sigma-Aldrich; ≥ 45 wt.-% SO ₃ according to manufacturer's information) and 1.21 g (3.14 mmol) of cholecalciferol (Sigma-Aldrich). The resulting solution was stirred vigorously for 1 h at 58 °C. Then 0.63 ml (0.456 g; 4.55 mmol) of triethylamine was added and stirring was continued for another 20 min at 58 °C.

The reaction mixture was then cooled in an ice bath at 0°C, 16.5 ml of cold methanol-trichloromethane (10:1 vol./vol.) solution was added, and stirring was continued for 20 min.

The solution was filtered through a glass frit and the solvent was removed by rotary evaporation. For further purification, the residue was treated twice with a methanol-trichloromethane (10:1 Vol./Vol.) solution and the solvent was removed by rotary evaporation, thereby obtaining 1.52 g (3.13 mmol; 99.7%) of cholecalciferol triethylammonium sulfate.

TLC (silica gel): R _F = 0.42 in methanol-trichloromethane (1:9 vol./vol.).

Example 2
Preparation of Cholecalciferol Ammonium Sulfate Approximately 14 ml of saturated ammonium acetate solution was added to 1.52 g (3.13 mmol) of triethylammonium cholecalciferol sulfate until a white precipitate of cholecalciferol ammonium sulfate was formed, which was left to stand overnight, then filtered using a glass frit and dried under high vacuum while cooling with tap water at a temperature of approximately 15° C.
Yield: 1.74 g (3.10 mmol, 99%).
Melting point: 104-108°C

Example 3
Preparation of cholecalciferol sodium sulfate

A saturated solution of sodium chloride (about 14 ml) was added to 1.52 g (3.13 mmol) of triethylammonium cholecalciferol sulfate until a white precipitate of sodium cholecalciferol sulfate was formed, which was left to stand overnight, then filtered using a glass frit and dried under high vacuum while cooling with tap water having a temperature of about 15° C.
Yield: 1.75 g (3.10 mmol, 99%).

The NMR spectrum was identical to that published by L. E. Reeve et al., The Journal of Biological Chemistry (1981), Vol. 256, No. 2, p. 824.

Example 4
Preparation of Cholecalciferol Magnesium Sulfate A saturated solution of magnesium chloride (about 13 ml) was added to 1.52 g (3.13 mmol) of triethylammonium cholecalciferol sulfate until a white precipitate of cholecalciferol magnesium sulfate was formed, which was left to stand overnight, then filtered using a glass frit and dried under high vacuum while cooling with tap water having a temperature of about 15° C.
Yield: 1.76 g (3.10 mmol; 99%).
Melting point: 107-110°C (decomposition).

Example 5
Preparation of cholecalciferol calcium sulfate
A saturated solution of _CaCl2 · _2H2O (about 13 ml) was added to 1.52 g (3.13 mmol) of triethylammonium cholecalciferol sulfate until a white precipitate of calcium cholecalciferol sulfate was formed, which was left to stand overnight, filtered using a glass frit, and dried under high vacuum while cooling with tap water having a temperature of about 15°C.
Yield: 1.81 g, (3.10 mmol, 99%).
TLC (silica gel): R _f = 0.48 in methanol-trichloromethane (1:9 Vol./Vol.)
Melting point: 97-101°C (decomposition).

Example 6
Preparation of cholecalciferol sulfate L-lysine

517.4 mg of pyridine-sulfur trioxide complex (Sigma-Aldrich; ≥ 45 wt.-% _SO3 according to manufacturer's information) (approximately 2.90 mmol of pyridine complexed with _SO3 ) and 506.3 mg (1.32 mmol) of cholecalciferol (Sigma-Aldrich) were dissolved in 4.4 ml of pyridine under nitrogen atmosphere and heated at 55 °C for 1 h with vigorous stirring. After adding 10 ml of ice-cold methanol-trichloromethane (1:9 Vol/Vol) mixture, the solvent was removed on a rotary evaporator. Then, 0.29 g (1.98 mmol) of L-lysine in 0.5 ml of sodium phosphate buffer, pH 7.3, was added and stirred for 5 min. Then, 20 ml of ice-cold methanol-trichloromethane (1:9 vol./vol.) mixture was added with stirring, after which the mixture was stripped on a rotary evaporator. 10 ml of absolute ethanol was added to the residue and the solution was stored in the refrigerator overnight. The ethanol was then decanted from the white creamy precipitate that formed and the residue was dried with cooling tap water at a temperature of about 15° C., thereby giving the title compound as a white powder (815 mg, (1.30 mmol); 98.5%).
TLC (silica gel): _Rf = 0.33 in methanol-trichloromethane (1:9 Vol./Vol.).
Melting point: 168℃ (decomposition)

NMR spectrum: see Figure 1

Example 7
Preparation of cholecalciferol sulfate D,L-lysine Cholecalciferol sulfate D,L-lysine was prepared in a manner similar to that of cholecalciferol sulfate L-lysine using DL-lysine (Sigma-Aldrich) instead of L-lysine.

The structure is confirmed by NMR spectroscopy in D ₄ -methanol.

Example 8
Transdermal formulation of cholecalciferol sulfate L-lysine in an oil base 100 mg of cholecalciferol sulfate L-lysine was mixed with 11.04 ml of oleic acid, then 0.83 ml of dimethyl sulfoxide was added and stirred with a magnetic stirrer at room temperature (21-25°C) for 2 days. Subsequently, 10 ml of glycerol trioleate and 7 ml of glycerol monooleate (Pecerol®, Gattefosse) were added and shaken vigorously. After waiting until the mixture had disappeared, an almost completely clear and stable solution of cholecalciferol sulfate L-lysine in the oil phase was obtained.

Example 9
Application of the transdermal formulation of Example 8 to the skin Approximately 2 ml of the formulation prepared in Example 8 was applied in a thin layer to the skin of the subject and allowed to penetrate for 4 hours. Subsequently, the skin was thoroughly wiped using a sterile cotton pad soaked in 96% ethanol. The cotton pad was boiled with additional 96% ethanol and squeezed. The ethanol was evaporated on a rotary evaporator and a small amount of chloroform-methanol (9:1 Vol./Vol.) mixture was added to the flask. TLC of this mixture on silica gel showed that virtually no cholecalciferol sulfate L-lysine (R _f :0.33) remained on the skin.

Example 10
Preparation of aqueous injection solutions
To prepare 2 ml of injection solution containing cholecalciferol sulfate in an amount corresponding to 10,000 IU of vitamin _D3 ,
a) 1.5695 mg of sodium cholecalciferol sulfate to produce injection solution A
b) 1.6243 mg of calcium cholecalciferol sulfate to produce injection solution B
and
c) 1.970 mg of cholecalciferol sulfate L-lysine to produce injection solution C
were each dissolved in 10 ml of distilled water. 89.9028 mg of sodium chloride was then added to each of the solutions to make them isotonic.

The pH of injection solution B was adjusted to pH 7 using 0.5N NaOH.

The solution was then filter-sterilized through a 0.22 μm membrane under an argon atmosphere and filled into 2 ml vials.

The disclosures of all documents cited herein, including, for example, patents, published patent applications, articles published in journals and books, are hereby incorporated by reference in their entirety.

Claims (1)

1. A pharmaceutical composition for combating vitamin D3 deficiency in a vertebrate animal comprising a pharma- ceutically acceptable salt of cholecalciferol sulfate, by a method of administration that allows at least 5% of said salt of cholecalciferol _sulfate to enter the systemic fluid transport system of the human body from the site of administration without being metabolized;
the method of administration is selected from transdermal and transmucosal administration, and intradermal, subcutaneous and intramuscular injections, including sustained drug release depot injections ;
A pharmaceutical composition, wherein the salt of cholecalciferol sulfate is cholecalciferol sulfate L-lysine .